scMAGeCK

In scMAGeCK: Identify genes associated with multiple expression phenotypes in single-cell CRISPR screening data

knitr::opts_chunk$set(echo = TRUE)

Introduction

scMAGeCK is a computational model to identify genes associated with multiple expression phenotypes from CRISPR screening coupled with single-cell RNA sequencing data (CROP-seq).

scMAGeCK is based on our previous MAGeCK and MAGeCK-VISPR models for pooled CRISPR screens, but further extends to scRNA-seq as the readout of the screening experiment. scMAGeCK consists of two modules: scMAGeCK-Robust Rank Aggregation (RRA), a sensitive and precise algorithm to detect genes whose perturbation links to one single marker expression; and scMAGeCK-LR, a linear-regression based approach that unravels the perturbation effects on thousands of gene expressions, especially from cells undergo multiple perturbations.

Usage

scmageck_rra

    library(scMAGeCK)
    ### BARCODE file contains cell identity information, generated from the cell identity collection step
    BARCODE <- system.file("extdata","barcode_rec.txt",package = "scMAGeCK")

    ### RDS can be a Seurat object or local RDS file path that contains the scRNA-seq dataset
    RDS <- system.file("extdata","singles_dox_mki67_v3.RDS",package = "scMAGeCK")

    ### Set RRA executable file path. 
    ###    You can generate RRA executable file by following commands:
    ###        wget https://bitbucket.org/weililab/scmageck/downloads/RRA_0.5.9.zip
    ###        unzip RRA_0.5.9.zip
    ###        cd RRA_0.5.9
    ###        make
    RRAPATH <- "/Library/RRA_0.5.9/bin/RRA"

    target_gene <- "MKI67"

    rra_result <- scmageck_rra(BARCODE=BARCODE, RDS=RDS, GENE=target_gene, RRAPATH=RRAPATH, 
             LABEL='dox_mki67', NEGCTRL=NULL, KEEPTMP=FALSE, PATHWAY=FALSE, SAVEPATH=NULL)

scmageck_lr

    library(scMAGeCK)
    ### BARCODE file contains cell identity information, generated from the cell identity collection step
    BARCODE <- system.file("extdata","barcode_rec.txt",package = "scMAGeCK")
    ### RDS can be a Seurat object or local RDS file path that contains the scRNA-seq dataset
    RDS <- system.file("extdata","singles_dox_mki67_v3.RDS",package = "scMAGeCK")

    lr_result <- scmageck_lr(BARCODE=BARCODE, RDS=RDS, LABEL='dox_scmageck_lr', 
            NEGCTRL = 'NonTargetingControlGuideForHuman', PERMUTATION = 1000, SAVEPATH=NULL, LAMBDA=0.01)
    lr_score <- lr_result[1]
    lr_score_pval <- lr_result[2]

Output

scmageck_rra

The scmageck_rra function will output the ranking and p values of each perturbed genes, using the RRA program in MAGeCK. Users familiar with the MAGeCK program may find it similar with the gene_summary output in MAGeCK.

Here is the example output of scMAGeCK-RRA:

Row.names  items_in_group.low  lo_value.low  p.low  FDR.low goodsgrna.low  items_in_group.high  lo_value.high  p.high  FDR.high  goodsgrna.high
TP53    271     0.11832 0.95619 1       48      271     1.014e-83       4.9975e-06      0.00015 184

Explanations of each column are below:

|Column|Content| |------|-------| |Row.names| Perturbed gene name| |items_in_group.low| The number of single-cells with each gene perturbed | |lo_value.low | The RRA score in negative selection (reducing the marker expression if this gene is perturbed). The RRA score uses a p value from rank order statistics to measure the degree of selection; the smaller score, the stronger the selection is. More information on the calculation of RRA score can be found in our original MAGeCK paper. |
|p.low | The raw p-value (using permutation) of this gene in negative selection |
|FDR.low | The false discovery rate of this gene in negative selection | |goodsgrna.low | The number of single-cells that passes the threshold and is considered in the RRA score calculation in negative selection| |items_in_group.high| The same as items_in_group.low: the number of single-cells with each gene perturbed) | |lo_value.high| The RRA score in positive selection (increasing the marker expression if this gene is perturbed|
|p.high| The raw p-value (using permutation) of this gene in positive selection |
|FDR.high| The false discovery rate of this gene in positive selection | |goodsgrna.high| The number of single-cells that passes the threshold and is considered in the RRA score calculation in positive selection|

scmageck_lr

The scmageck_lr function will generate several files below:

|File|Description| |----|----------| |lr_score|The score (similar with log fold change) of each perturbed gene (rows) on each marker gene (columns)| |lr_score.pval|The associated p values of each score| |LR.RData|An R object to store scores and p values|

The format of score.txt and score.pval.txt is a simple table file with rows corresponding to perturbed genes and columns corresponding to marker genes. For example in the score.txt,

Perturbedgene  APC                ARID1A               TP53               MKI67
     APC       0.138075836476524  -0.0343441660045313  0.214449590551132  -0.150287676553705

This row records the effects of perturbing APC gene on the expressions of APC, ARID1A, TP53 and MKI67.

Contact us

Questions? Comments? Join the MAGeCK Google group or email us ([e-mail]) directly.

Any advice and suggestions will be greatly appreciated.

scMAGeCK documentation built on Nov. 8, 2020, 7:49 p.m.